The goal of this project is to elucidate structure-function relationships in macromolecular machines. During FY11, we studied: (1) energy-dependent proteases involved in protein quality control and cell regulation;(2) membrane remodelling;and (3) a DNA transposition system. (1) All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute them. Programmed degradation of regulatory factors also contributes to controlling the cell cycle and to generating peptides for immune presentation. These activities are all carried out by energy-dependent proteolytic machines, which generically consist of two subcomplexes - a protase and a chaperone-like ATPase. For several years, we have studied the Clp complexes of E. coli, considered as a model system. We showed that the protease ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. ClpA/X stack axially on one or both faces of ClpP in active complexes. We went on to show that substrate proteins bind to distal sites on the ATPase and are then unfolded and translocated axially into the digestion chamber of ClpP. In FY11, we completed the publication of two papers (1, 2) reporting work performed in FY10. In one, we reconstructed the ClpA hexamer as an integral part of the ClpAP complex. Two protein segments lining the axial channel were found to exhibit local mobility. We also found that ATP hydrolysis is accompanied by substantial structural changes in the D2 tier but not in the D1 tier of ClpA and confirmed that Its entire N-domain undergoes large-scale fluctuations that render it invisible in averaged electron micrographs. The second paper addressed gating of the axial pore in ClpP. In the absence of ClpA, the pore is closed but it opens up on ClpA binding, creating an access channel. This region is occupied by the N-terminal loops of ClpP. Thus access to the ClpP degradation chamber is controlled by hinged movements of its N-terminal loops. &#8232;&#8232;In FY11, we initiated EM studies addressing the heteromeric ClpP found in chloroplasts. The differing subunits in this assembly contrast with the ClpP tetradecamer and the heptamer of human mitochondria, which are homomeric, possibly indicating the presence of proteolytic active sites with multiple specificities. (2) Membrane Remodelling. Remodelling, a process in which lipid bilayer structures are reconfigured by interacting proteins, is central to the functioning and metabolism of cells. We are investigating this phenomenon by using cryo-electron microscopy to characterize the effects of remodelling proteins such as endophilin and alpha-synuclein on large lipid vesicles in vitro. Endophilin A1 is a BAR (Bin/Amphiphysin /Rvs) protein abundant in neural synapses that senses and induces membrane curvature, contributing to neck formation in pre-synaptic endocytic vesicles. We found that, on exposure to endophilin, vesicles convert rapidly to coated tubules whose morphology reflects the local concentration of endophilin. Their diameters and curvature resemble those of synaptic vesicles in situ. 3D reconstructions of quasi-cylindrical tubes revealed arrays of BAR dimers, flanked by densities that we equate with amphipathic. We also observed the compression of bulbous coated tubes into 70-wide cylindrical micelles, which appear to mimic the penultimate (hemi-fission) stage of endocytosis. Our findings suggest that the adaptability of endophilin-lipid interactions underlies dynamic changes of endocytic membranes (3).&#8232;In FY11, this project has been extended to the protein, a-synucleins (aS) (4). Natively unfolded in solution, aS accumulates as amyloid in neurological tissue in Parkinsons disease, and also interacts with membranes under both physiological and pathological conditions. Its interaction with lipids is mediated by motifs that form amphipathic helices. We have used cryo-electron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the effect of aS on lipid vesicles. The products obtained depend on the protein : lipid ratio. At a molar ratio of <1: 40, POPG vesicles are converted into cylindrical micelles 50 in diameter, together with bilayer tubes of various widths (150 - 500 ). Between 1 : 20 and 1 : 10, cylindrical micelles are produced exclusively. Other negatively charged lipids (DMPG, DLPG, DAPG) exhibit generally similar behavior. At higher protein: lipid ratios (>1: 10), cylindrical micelles are replaced by discoid particles, 70 - 100 across. These observations are being prepared for publication. 3) In DNA transposition, segments of chromosomal DNA are reshuffled. The lysogenic bacteriophage Mu affords a tractable model system to study this process. The proteins MuA and MuB are required for integration and amplification of the Mu genome. MuA plays a direct role in the transposition reaction, while MuB stimulates MuA activity and selects target sites on the host chromosome. From sequence analysis, we have identified MuB as an AAA+-like protein, consisting of an N-terminal (res. 1-60) DNA-binding domain (NTD) and a C-terminal AAA+ domain (res. 61 - 311). Using electron microscopy, we found that MuB forms filaments 150 in diameter in the presence of ATP but not in the presence of ADP. The presence of DNA enhances filament formation. We used cryo-EM and image reconstruction to investigate filaments formed with and without DNA. in both cases, the filament is a 1-start helix with a helical pitch of 47 and the number of subunits per turn varying between 5.6 and 6.2. As a truncated mutant lacking the NTD also forms filaments, it follows that the main body of the solenoid is a strand of AAA+ domains. The NTDs are connected to it by a flexible linker. In the absence of DNA, a significant population of NTDs accumulate in the axial region of filament (closed conformation): in the presence of DNA, no such density is detected and the solenoid is open to accommodate DNA (open conformation). Combining these data with the earlier observation that interaction with MuA stimulates MuB ATPase activity, our data support the following model: MuB-ATP targets the DNA by making a nucleoprotein filament, and upon interacting with MuA, MuB hydrolyzes ATP, the MuB-DNA filament partially disassembles, and a segment of DNA is exposed for the action of MuA. A paper reporting this study is almost ready for submission.